E Novo Dna Methylation Programs Regulate Car T-Cell Exhaustion

Abstract Chimeric antigen receptor (CAR) T-cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies. While clinical trials have demonstrated the curative potential of this approach, a significant and well-established limitation is the heightened contraction and transient persistence that CAR T-cells experience during prolonged antigen exposure. Building upon our prior observation that deleting the de novo DNA methyltransferase 3a (Dnmt3a) prevents T cell exhaustion in the prototypical model of LCMV-induced T cell exhaustion, we assessed the role of DNMT3A programming in the dysfunction of human CAR T-cells. Deletion of DNMT3A in multiple human CAR T-cell systems resulted in a striking preservation of the CAR T-cell’s ability to proliferate and mount an effector response during chronic antigen exposure. Whole genome methylation profiling of DNMT3A KO CAR T-cells established an atlas of epigenetically regulated genes targeted during CAR T-cell dysfunction. Cross-reference of our published murine exhaustion methylation profiles with our newly identified human CAR T-cell methylation atlas revealed conservation of epigenetically regulated exhaustion-associated genes. Using a novel epigenetic-based bioinformatic tool that predicts human T-cell differentiation we further documented the preserved developmental plasticity of the DNMT3A KO CAR T-cells. Lastly, analysis of publicly available RNAseq expression data from CD19-CAR T-cell products prior to infusion into CLL patients demonstrated that DNMT3A programming is significantly coupled to clinical outcome. Collectively our results demonstrate that de novo DNA methylation programming is a key factor limiting T-cell based immunotherapy.